Focal expression of mutant huntingtin in the songbird basal ganglia disrupts cortico-basal ganglia networks and vocal sequences

The basal ganglia (BG) promote complex sequential movements by helping to select elementary motor gestures appropriate to a given behavioral context. Indeed, Huntington’s disease (HD), which causes striatal atrophy in the BG, is characterized by hyperkinesia and chorea. How striatal cell loss alters activity in the BG and downstream motor cortical regions to cause these disorganized movements remains unknown. Here, we show that expressing the genetic mutation that causes HD in a song-related region of the songbird BG destabilizes syllable sequences and increases overall vocal activity, but leave the structure of individual syllables intact. These behavioral changes are paralleled by the selective loss of striatal neurons and reduction of inhibitory synapses on pallidal neurons that serve as the BG output. Chronic recordings in singing birds revealed disrupted temporal patterns of activity in pallidal neurons and downstream cortical neurons. Moreover, reversible inactivation of the cortical neurons rescued the disorganized vocal sequences in transfected birds. These findings shed light on a key role of temporal patterns of cortico-BG activity in the regulation of complex motor sequences and show how a genetic mutation alters cortico-BG networks to cause disorganized movements.The execution of complex behaviors, such as speech or musicianship, depends on the ability to organize elementary motor “gestures” into precisely timed sequences of movements. In the vertebrate brain, the basal ganglia (BG) play a key role in regulating complex motor sequences, as revealed by the behavioral disruptions characteristic of neurological diseases that affect BG function (1–3). A particularly striking example is Huntington’s disease (HD), which is characterized anatomically by extensive cell death in the striatum, the primary input layer of the BG network (4), and behaviorally by hyperkinesia and chorea, which can interfere with the ability to produce coordinated, sequential movements (5, 6). Despite numerous animal models of HD, the detailed neural mechanisms by which striatal dysfunction leads to disorganized movements have remained elusive. One challenge is that HD also affects brain regions other than the BG, making it difficult to identify causal links between BG circuit dysfunction and behavioral symptoms. Another challenge, especially in rodent models of HD, is the limited understanding of whether and how BG circuitry contributes to specific behaviors.The zebra finch is a small Australian songbird that presents several advantages for addressing these challenges. Adult male zebra finches sing a learned song comprising a reproducible sequence of stereotyped syllables (i.e., a motif), and they sing both spontaneously and abundantly, allowing for extensive characterization of syllables and syllable sequences. Moreover, an anatomically differentiated network of interconnected brain nuclei important to singing distinguishes the zebra finch brain (7, 8). An anterior forebrain pathway within this song system shares many similarities to mammalian cortico-BG networks (Fig. 1A), including a BG nucleus (area X) that contains both medium spiny neurons (MSNs) and pallidal neurons, which closely resemble their mammalian counterparts in their intrinsic, synaptic, and circuit properties (9–13). Pallidal neurons in area X communicate via the thalamus to a cortical premotor nucleus [lateral magnocellular nucleus of the anterior nidopallium (LMAN)] that plays a critical role in modulating song variability (14–18). The dedicated nature of this pathway for singing provides a rare opportunity to explore a detailed mechanism by which deficits in the BG act across broader cortico-BG networks to cause specific behavioral symptoms. Furthermore, the spatially distributed organization of the component song nuclei is well suited to the use of viral methods for selectively expressing genetic constructs within a single node in this network (19–22).Open in a separate windowFig. 1.Expression of mHTT in the songbird BG. (A) Simplified schematic showing some of the common features of mammalian and songbird cortico-BG networks. (B) Time line of experiments (Top) and a schematic showing a part of the neural circuitry involved in singing behaviors in the songbird (Bottom; sagittal view with rostral to the right and dorsal upward). LV containing a mutant form of mHTT and a fluorescent reporter, AsRed, with insertion of a self-cleaving T2A sequence was injected into the songbird BG (area X) of adult male zebra finches. The songs before injection (habituation) were highly stable (Fig. S1). Behavioral or neural changes were assessed at >30 d after injection. dph, days posthatch. (C) Square region in area X in a sagittal brain slice (Top; D, dorsal; R, rostral) was imaged 34 d after injection with a confocal microscope (Bottom) to confirm expression of mHTT-Q94, as revealed by coexpressed AsRed (see also Fig. S1).Intriguingly, a transgenic zebra finch model of HD was recently generated that shares several similarities with human HD (23), lending support to the idea that songbirds can afford useful models in which to identify how genetic mutations that cause HD alter BG circuitry to affect behavior. Indeed, these transgenic HD songbirds showed deficits in song learning; produced songs with abnormal syllable and sequence variability as adults; and displayed other symptoms, including body rigidity, tremor, and cervical dystonia. However, as with transgenic rodent models of HD and patients with HD, the brain-wide expression of the genetic mutation that causes HD in these transgenic songbirds makes it challenging to understand the link between genotype, circuit pathology, and behavioral phenotype. Here, we overcame this challenge by virally expressing a mutant gene fragment that causes HD [exon 1 of the huntingtin gene (mHTT)] in area X of adult male zebra finches. This focal genetic perturbation caused selective deficits in vocalization: The transfected birds vocalized more abundantly and sang aberrant songs in which syllable sequences, but not individual syllables, were selectively destabilized. These vocal changes were accompanied by the selective loss of MSNs in area X, a marked reduction of inhibitory synapses on pallidal neurons, and disrupted temporal patterns of singing-related activity in pallidal and LMAN neurons. Moreover, reversibly inactivating LMAN was sufficient to restore normal vocal behavior. These findings indicate that focal expression of mHTT in the BG disrupts motor sequences by altering temporal patterns of cortico-BG activity, which may afford a novel target for ameliorating disorganized movements in BG diseases.     

Accelerated expansion of pathogenic mitochondrial DNA heteroplasmies in Huntington’s disease

Mitochondrial dysfunction is found in the brain and peripheral tissues of patients diagnosed with Huntington’s disease (HD), an irreversible neurodegenerative disease of which aging is a major risk factor. Mitochondrial function is encoded by not only nuclear DNA but also DNA within mitochondria (mtDNA). Expansion of mtDNA heteroplasmies (coexistence of mutated and wild-type mtDNA) can contribute to age-related decline of mitochondrial function but has not been systematically investigated in HD. Here, by using a sensitive mtDNA-targeted sequencing method, we studied mtDNA heteroplasmies in lymphoblasts and longitudinal blood samples of HD patients. We found a significant increase in the fraction of mtDNA heteroplasmies with predicted pathogenicity in lymphoblasts from 1,549 HD patients relative to lymphoblasts from 182 healthy individuals. The increased fraction of pathogenic mtDNA heteroplasmies in HD lymphoblasts also correlated with advancing HD stages and worsened disease severity measured by HD motor function, cognitive function, and functional capacity. Of note, elongated CAG repeats in HTT promoted age-dependent expansion of pathogenic mtDNA heteroplasmies in HD lymphoblasts. We then confirmed in longitudinal blood samples of 169 HD patients that expansion of pathogenic mtDNA heteroplasmies was correlated with decline in functional capacity and exacerbation of HD motor and cognitive functions during a median follow-up of 6 y. The results of our study indicate accelerated decline of mtDNA quality in HD, and highlight monitoring mtDNA heteroplasmies longitudinally as a way to investigate the progressive decline of mitochondrial function in aging and age-related diseases.

Huntington’s disease (HD) is a monogenic disorder caused by the expansion of cytosine–adenine–guanine trinucleotide (CAG) repeats in the HTT gene at chromosome 4p16.3 (1). Although HTT is expressed in various tissues, the brain, particularly the striatum, is vulnerable to mutant huntingtin (mHTT)-associated toxicity (2). The average age at onset of the characteristic motor symptoms of HD is between 40 and 50 y old, followed by a progressive decline of motor, cognitive, and psychiatric functions for an average of 20 y prior to death (3).The biological processes that determine the onset and progression of HD are still elusive. Recent studies suggest that mitochondrial dysfunction may be involved in HD pathogenesis (4, 5). Mitochondria are subcellular organelles of eukaryotes, which play vital roles in maintaining energetic and metabolic homeostasis (6, 7). Evidence for mitochondrial dysfunction in HD was first reported in the postmortem brain of HD patients, which show low mitochondrial oxidative phosphorylation (OXPHOS) protein activity and energy deficits (8–10). Mitochondrial dysfunction was further found in peripheral tissues and cell lines of HD patients, such as blood, lymphoblasts, skeletal muscle, and skin fibroblasts (11–17).Several molecular mechanisms have been proposed to connect mHTT to mitochondrial dysfunction. Studies in HD knockin mice indicate that toxic fragments derived from mHTT can suppress mitochondrial biogenesis and energy metabolism (18). mHTT has also been found to physically interact with mitochondria, reducing mitochondrial membrane potential (13, 19). Furthermore, mHTT may stimulate mitochondrial network fragmentation (20–22), and it has recently been found to impair mitophagy (23–28), an evolutionarily conserved quality control system in eukaryotes to selectively remove dysfunctional mitochondria (29). Perturbation of mitochondrial tubular networks, morphology, and mitophagy are pathological features common to various neurodegenerative diseases (30, 31).Mitochondrial function is determined not only by the nuclear genome but also by the mitochondrial genome (mtDNA). Human mtDNA is a 16.6-kb circular DNA located within mitochondria. It encodes 13 evolutionarily conserved proteins in four of the five OXPHOS protein complexes (32). The accumulation of mtDNA mutations in somatic tissues has been suggested as a possible driver of age-related mitochondrial dysfunction (33). Transgenic mice with an increased level of mtDNA mutations manifest progeroid phenotypes and early neurodegeneration that resemble human aging (34, 35). Clonal expansion of preexisting mtDNA mutations in somatic tissues has also been shown to contribute to accelerated mitochondrial aging and OXPHOS defects in human diseases (36, 37).Because there are multiple copies of mtDNA in a single cell, mutations can arise and coexist with wild-type mtDNA in a state called heteroplasmy, which has been linked to a variety of mitochondrial disorders in humans (32, 38). Our previous study on lymphoblasts from the 1,000 Genomes project indicates that about 90% of individuals in the general population carry at least one heteroplasmy in mtDNA, and purifying selection keeps most pathogenic heteroplasmies at a low fraction (39). Thus, when such a selective constraint on mitochondria is weakened under certain conditions (40), such as the presence of mHTT (20–28), these low-fraction pathogenic heteroplasmies may increase in their fractions in cells, culminating in dysfunctional mitochondria and related energy deficits (32).In the current study, we hypothesized that HD progression is partially driven by the deterioration of mtDNA quality. Since HTT is universally expressed, and mitochondrial dysfunction has been repeatedly observed in peripheral tissues (11–16), we surmised that HD-associated mtDNA changes can be detected in peripheral tissues and cell lines of HD patients, such as blood-derived lymphoblasts, which are readily available in large patient cohorts and thus can provide increased power for identifying mtDNA changes in HD. To test this hypothesis, we employed a sensitive mtDNA targeted sequencing approach, STAMP (sequencing by targeted amplification of multiplex probes) (41), to assess mtDNA heteroplasmies in lymphoblast and longitudinal blood samples from HD patients and healthy control individuals in the European Huntington’s Disease Network’s REGISTRY study (hereafter referred to as REGISTRY) (42). We achieved ultradeep sequencing coverage on mtDNA in these samples and revealed an accelerated expansion of pathogenic mitochondrial DNA heteroplasmies in HD, illustrating a molecular feature underlying HD biology.     

Amelioration of pathologic α-synuclein-induced Parkinson’s disease by irisin

Physical activity provides clinical benefit in Parkinson’s disease (PD). Irisin is an exercise-induced polypeptide secreted by skeletal muscle that crosses the blood–brain barrier and mediates certain effects of exercise. Here, we show that irisin prevents pathologic α-synuclein (α-syn)-induced neurodegeneration in the α-syn preformed fibril (PFF) mouse model of sporadic PD. Intravenous delivery of irisin via viral vectors following the stereotaxic intrastriatal injection of α-syn PFF cause a reduction in the formation of pathologic α-syn and prevented the loss of dopamine neurons and lowering of striatal dopamine. Irisin also substantially reduced the α-syn PFF-induced motor deficits as assessed behaviorally by the pole and grip strength test. Recombinant sustained irisin treatment of primary cortical neurons attenuated α-syn PFF toxicity by reducing the formation of phosphorylated serine 129 of α-syn and neuronal cell death. Tandem mass spectrometry and biochemical analysis revealed that irisin reduced pathologic α-syn by enhancing endolysosomal degradation of pathologic α-syn. Our findings highlight the potential for therapeutic disease modification of irisin in PD.

Parkinson’s disease (PD) is a chronic neurodegenerative disorder characterized by progressive worsening of motor symptoms, including bradykinesia, resting tremor, and rigidity (1, 2). Nonmotor symptoms often precede and accompany the motor symptoms and include autonomic dysfunction and neuropsychiatric sequelae (3). The most notable loss of neurons occurs in the dopaminergic neurons of the substantia nigra pars compacta (SNpc), although neuronal loss also occurs in the locus coeruleus, dorsal raphe nucleus, the dorsal motor nucleus of the vagus, and nucleus basalis of Meynert (4). In addition to neuronal loss, there is accumulation of misfolded pathologic α-synuclein that drives the pathogenesis of PD, including the neuronal dysfunction and the ultimate of neuronal degeneration (5, 6). Current treatments for PD include the replacement of dopamine (DA) via L-DOPA, DA agonists, and other agents to treat the nonmotor symptoms. As the disease progresses, deep brain stimulation and other neurosurgical approaches can be used to treat the side effects of DA replacement therapy. Importantly, these treatments only address the symptomology, and over time there is a progressive decline in normal function. Moreover, there are no treatments that slow the progression or inhibit the underlying drivers of PD pathogenesis. As such, treatments that result in durable arrest of PD symptoms are urgently needed.Irisin is a small polypeptide that is secreted by skeletal muscle and other tissues into the blood of mice and humans (7, 8). The amino acid sequence is conserved 100% between mice and humans, suggesting a critical, conserved function. Importantly, the expression of irisin and its precursor protein FNDC5 is increased in muscle in response to many forms of exercise, both in rodents and in humans. Irisin levels increase in the blood of humans with exercise training, as determined by tandem mass spectrometry (8). In adipose cells, osteocytes, osteoclasts, and astrocytes integrin αV/β5 is the major functioning receptor for irisin (9, 10).Physical activity can prevent and ameliorate the symptoms of multiple forms of neurodegeneration, including Alzheimer’s disease (AD) and PD (11–14). Since irisin carries some of the benefits of exercise to adipose tissues, we and others have begun to study the effects of irisin in various models of neurodegeneration. In the earliest study, we showed that elevated expression of FNDC5 in the liver via the use of adenoviral vectors, and presumptive elevations of irisin in the blood, stimulated an “exercise-like” program of gene expression in the hippocampus (15). Moreover, the expression of FNDC5 with these same viral vectors rescued memory deficits in a mouse model of AD (16). Most recently, irisin itself was shown to be the active moiety regulating cognitive function in four separate mouse models. Importantly, elevation of the blood levels of the mature, cleaved irisin using adeno-associated virus (AAV) was sufficient to improve cognitive function and reduce neuroinflammation in two distinct models of AD (9). Furthermore, irisin itself crossed the blood–brain barrier (BBB), at least when the protein was produced from the liver with these AAV vectors.In the current study, we examine the effects of irisin on the pathophysiology of PD, using the α-synuclein preformed fibril (α-syn PFF) seeding model in vitro and in vivo. Pathologic α-syn is thought to spread “prion-like” in the brains of PD patients and certain other neurological disorders, where they cause neuronal death and dysfunction. We show here that irisin has powerful effects in preventing both the accumulation of pathologic α-syn and neuronal cell death in primary cell culture. Furthermore, elevation of blood irisin levels in mice normalizes the histological manifestations in the SNpc and the PD-like symptomology involving movement and grip strength induced by intrastriatal injection of α-syn PFF. Together, these data suggest the potential therapeutic value of irisin in PD and other neurodegenerative states that involve α-syn.     

Transcellular spreading of huntingtin aggregates in the Drosophila brain

A key feature of many neurodegenerative diseases is the accumulation and subsequent aggregation of misfolded proteins. Recent studies have highlighted the transcellular propagation of protein aggregates in several major neurodegenerative diseases, although the precise mechanisms underlying this spreading and how it relates to disease pathology remain unclear. Here we use a polyglutamine-expanded form of human huntingtin (Htt) with a fluorescent tag to monitor the spreading of aggregates in the Drosophila brain in a model of Huntington’s disease. Upon expression of this construct in a defined subset of neurons, we demonstrate that protein aggregates accumulate at synaptic terminals and progressively spread throughout the brain. These aggregates are internalized and accumulate within other neurons. We show that Htt aggregates cause non–cell-autonomous pathology, including loss of vulnerable neurons that can be prevented by inhibiting endocytosis in these neurons. Finally we show that the release of aggregates requires N-ethylmalemide–sensitive fusion protein 1, demonstrating that active release and uptake of Htt aggregates are important elements of spreading and disease progression.Accumulation of protein aggregates is a key feature of many neurodegenerative diseases. Lesions in each of these diseases are initially limited to defined regions of selectively vulnerable neurons, but staging of pathology in Alzheimer’s disease (1), Parkinson’s disease (2, 3), amyotropic lateral sclerosis (4, 5), and Huntington’s disease (HD) (6) reveal broader deposition of pathological aggregates at more advanced stages of disease progression. The observation that the pathology appeared to progress into regions that were synaptically connected led to the idea that pathology was spreading through neuronal circuits (7, 8). Converging lines of evidence demonstrated that aggregates of disease-associated misfolded proteins, including α-synuclein, tau, and superoxide dismutase 1, are in fact transmissible from cell to cell and that this transmission propagates throughout the brain (9, 10). More recently, mutant huntingtin (Htt) aggregates were also shown to spread between neurons in vivo (11).Although the cell-to-cell spreading of pathogenic proteins has been demonstrated in several neurodegenerative diseases, the mechanism by which this spreading occurs, and how it contributes to pathology and later stages of disease progression, remain unclear. To gain a better understanding of how this protein spreading contributes to disease pathology, we sought to study this phenomenon in Drosophila. Drosophila has been used to create useful models of many neurodegenerative diseases, including Parkinson’s disease (12) and the polyglutamine (polyQ) expansion diseases spinocerebellar ataxia type 1 (13) and type 3 (14) as well as Huntington’s disease (15–18). These models have proven to reproduce many of the key structural and functional deficits associated with disease pathology and provide insight into the underlying mechanisms. For example, a recent study demonstrated a “prion-like” spread of huntingtin aggregates into phagocytic glia, cells which carry out a protective clearance function but also potentially contribute to spreading itself (19). One advantage to studying protein aggregate spreading in Drosophila is the ability to independently label and manipulate separate populations of neurons simultaneously by using the yeast Gal4/Upstream Activating Sequence (UAS) (20) and bacterial LexA/LexA operator (LexAop) (21) binary expression systems. Additionally, the ability to rapidly identify and characterize genetic and chemical modifiers of this spreading phenomenon should help unravel mechanisms responsible for spreading.In this study, we demonstrate that mutant huntingtin aggregates accumulate at synaptic terminals in the antennal lobe of the Drosophila central brain when expressed in olfactory receptor neurons (ORNs). Over time, these aggregates begin to spread to various regions of the brain, where they are internalized by other populations of neurons, resulting in some instances in loss of these neurons. This neuronal loss is prevented by blocking endocytosis, suggesting that spreading requires active internalization of the pathogenic protein. We observe unique spreading patterns when huntingtin is expressed in different populations of neurons, supporting the idea that nearby cells and neuronal circuits are likely targets of spreading. However, rapid accumulation of aggregates far from the original source also suggests that transmission is not limited to these circuits. The release of aggregates depends on N-ethylmaleimide–sensitive fusion protein 1 (NSF1), suggesting that soluble NSF attachment protein receptor (SNARE)-mediated fusion events are required for aggregate spreading. The extensive and efficient spread of huntingtin aggregates in the Drosophila brain that we report here provides a powerful experimental system for detailed genetic, molecular, and cellular analyses to dissect the underlying mechanisms and consequences.     

Regulation of beta-amyloid production in neurons by astrocyte-derived cholesterol

Alzheimer’s disease (AD) is characterized by the presence of amyloid β (Aβ) plaques, tau tangles, inflammation, and loss of cognitive function. Genetic variation in a cholesterol transport protein, apolipoprotein E (apoE), is the most common genetic risk factor for sporadic AD. In vitro evidence suggests that apoE links to Aβ production through nanoscale lipid compartments (lipid clusters), but its regulation in vivo is unclear. Here, we use superresolution imaging in the mouse brain to show that apoE utilizes astrocyte-derived cholesterol to specifically traffic neuronal amyloid precursor protein (APP) in and out of lipid clusters, where it interacts with β- and γ-secretases to generate Aβ-peptide. We find that the targeted deletion of astrocyte cholesterol synthesis robustly reduces amyloid and tau burden in a mouse model of AD. Treatment with cholesterol-free apoE or knockdown of cholesterol synthesis in astrocytes decreases cholesterol levels in cultured neurons and causes APP to traffic out of lipid clusters, where it interacts with α-secretase and gives rise to soluble APP-α (sAPP-α), a neuronal protective product of APP. Changes in cellular cholesterol have no effect on α-, β-, and γ-secretase trafficking, suggesting that the ratio of Aβ to sAPP-α is regulated by the trafficking of the substrate, not the enzymes. We conclude that cholesterol is kept low in neurons, which inhibits Aβ accumulation and enables the astrocyte regulation of Aβ accumulation by cholesterol signaling.

Alzheimer’s disease (AD), the most prevalent neurodegenerative disorder, is characterized by the progressive loss of cognitive function and the accumulation of amyloid β (Aβ) peptide and phosphorylated tau (1). Amyloid plaques are composed of aggregates of Aβ peptide, a small hydrophobic protein excised from the transmembrane domain of amyloid precursor protein (APP) by proteases known as beta- (β-) and gamma- (γ-) secretases (SI Appendix, Fig. S1A). In high concentrations, Aβ peptide can aggregate to form Aβ plaques (2–4). The nonamyloidogenic pathway involves a third enzyme, alpha- (α-) secretase, which generates a soluble APP fragment (sAPP-α), helps set neuronal excitability in healthy individuals (5), and does not contribute to the generation of amyloid plaques. Therefore, by preventing Aβ production, α-secretase–mediated APP cleavage reduces plaque formation. Strikingly, both pathways are finely regulated by cholesterol (6) (SI Appendix, Fig. S1B).In cellular membranes, cholesterol regulates the formation of lipid clusters (also known as lipid rafts) and the affinity of proteins to lipid clusters (7), including β-secretase and γ-secretase (8–10). α-secretase does not reside in lipid clusters; rather, α-secretase is thought to reside in a region made up of disordered polyunsaturated lipids (11). The location of APP is less clear. In detergent-resistant membrane (DRM) studies, it primarily associates with lipid from the disordered region, although not exclusively (8, 10, 12–14). Endocytosis is thought to bring APP in proximity to β-secretase and γ-secretase, and this correlates with Aβ production. Cross-linking of APP with β-secretase on the plasma membrane also increases Aβ production, leading to a hypothesis that lipid clustering in the membrane contributes to APP processing (11, 14, 15) (SI Appendix, Fig. S1A). Testing this hypothesis in vivo has been hampered by the small size and transient nature of lipid clusters (often <100 nm), which is below the resolution of light microscopy.Superresolution imaging has emerged as a complimentary technique to DRMs, with the potential to interrogate cluster affinity more directly in a native cellular environment (16). We recently employed superresolution imaging to establish a membrane-mediated mechanism of general anesthesia (17). In that mechanism, cholesterol causes lipid clusters to sequester an enzyme away from its substrate. Removal of cholesterol then releases and activates the enzyme by giving it access to its substrate (SI Appendix, Fig. S1C) (7, 18). A similar mechanism has been proposed to regulate the exposure of APP to its cutting enzymes (11, 15, 19–21).Neurons are believed to be the major source of Aβ in normal and AD brains (22, 23). In the adult brain, the ability of neurons to produce cholesterol is impaired (24). Instead, astrocytes make cholesterol and transport it to neurons with apolipoprotein E (apoE) (25–27). Interestingly, apoE, specifically the e4 subtype (apoE4), is the strongest genetic risk factor associated with sporadic AD (28, 29). This led to the theory that astrocytes may be controlling Aβ accumulation through regulation of the lipid cluster function (11, 15, 19), but this has not yet been shown in the brain of an animal. Here, we show that astrocyte-derived cholesterol controls Aβ accumulation in vivo and links apoE, Aβ, and plaque formation to a single molecular pathway.     

Synthetic lethal screening in the mammalian central nervous system identifies Gpx6 as a modulator of Huntington’s disease

Huntington’s disease, the most common inherited neurodegenerative disease, is characterized by a dramatic loss of deep-layer cortical and striatal neurons, as well as morbidity in midlife. Human genetic studies led to the identification of the causative gene, huntingtin. Recent genomic advances have also led to the identification of hundreds of potential interacting partners for huntingtin protein and many hypotheses as to the molecular mechanisms whereby mutant huntingtin leads to cellular dysfunction and death. However, the multitude of possible interacting partners and cellular pathways affected by mutant huntingtin has complicated efforts to understand the etiology of this disease, and to date no curative therapeutic exists. To address the general problem of identifying the disease-phenotype contributing genes from a large number of correlative studies, here we develop a synthetic lethal screening methodology for the mammalian central nervous system, called SLIC, for synthetic lethal in the central nervous system. Applying SLIC to the study of Huntington’s disease, we identify the age-regulated glutathione peroxidase 6 (Gpx6) gene as a modulator of mutant huntingtin toxicity and show that overexpression of Gpx6 can dramatically alleviate both behavioral and molecular phenotypes associated with a mouse model of Huntington’s disease. SLIC can, in principle, be used in the study of any neurodegenerative disease for which a mouse model exists, promising to reveal modulators of neurodegenerative disease in an unbiased fashion, akin to screens in simpler model organisms.All of the major neurodegenerative diseases display characteristic nerve-cell (neuronal) vulnerability patterns, as well as an increased prevalence with advanced age. Due to the latter, it has been reasoned that knowledge of the normal aging-associated gene expression changes in vulnerable neuronal populations would reveal pathways that intersect with disease-associated molecular mechanisms. Numerous studies have examined age-related gene expression changes in human and rodent brain, revealing altered expression of genes involved in many cellular processes, including synaptic plasticity, mitochondrial function, proteasomal function, antioxidant responses, as well as DNA damage repair (1–4). However, these studies have focused almost exclusively on gene expression changes averaged over entire brain regions without resolving the multiple distinct neuronal cell types within these regions. To study normal age-associated molecular pathways in individual neurodegenerative disease-relevant cell types in situ, we used the translating ribosome affinity purification (TRAP) methodology (5, 6) to create cell-type–specific molecular profiles of cell populations during normal mouse brain aging. Due to their vulnerability in Huntington’s disease (HD), we profiled dopaminoceptive neurons in the striatum and cortex, as defined by cells that express the Drd1a or Drd2 dopamine receptors at 6 wk and 2 y of age (all research involving vertebrate animals was approved by the MIT institutional review board). Each cell type displayed a unique pattern of gene expression changes associated with aging that were consistent across technical replicates and across mice (Datasets S1–S4 and Fig. 1). Only five genes, including two predicted pseudogenes, displayed a statistically significant altered expression with aging in all cell types (Tnnt2, Gm5425, Rnd3, Pisd, and Pisd-ps3), indicating that there is not a common aging program across the cell types studied, but rather that even closely related cell types show distinct molecular changes during normal aging. Pathways analysis of age-regulated genes revealed several molecular pathways altered by aging in each cell type (Datasets S5–S8). In Drd2-expressing striatal neurons, which displayed the greatest number of altered gene pathways during aging, “glutathione-mediated detoxification” and “glutathione redox reactions” were among the top gene pathways altered with age (including the genes Gsta3, Gsta4, Gstm1, Gstm6, Gpx1, Gpx2, and Gpx6). Oxidative damage has long been linked to aging (7). Given that oxidative damage to DNA, proteins, and lipids has been reported to increase with age in the brain (8–10), the increases to glutathione-dependent enzymes that we report here likely reflect a homeostatic neuronal response to increased oxidative damage in this cell population.Open in a separate windowFig. 1.Gene expression changes associated with normal aging in cortical and striatal dopaminoceptive cell types. Venn diagram showing the number and overlap of statistically significant gene expression changes in dopamine receptor 1a (D1)- or dopamine receptor 2 (D2)-expressing cortical or striatal neurons, based on a comparison of mice aged 6 wk of age versus 2 y of age. Statistically significant changes are defined as genes displaying ≥1.2-fold change and a Benjamini–Hochberg adjusted P value from Welch’s t test of ≤0.05.Although these experiments identified a promising set of age-regulated candidate genes that may be relevant to the age-associated progression of Huntington’s disease, they did not single out any one factor as contributing above others. In addition, the large aggregate number of changes observed in these cell types prevented us from inspecting the effects of each gene expression change on Huntington’s disease progression with traditional means such as mouse knockout or overexpression studies. To address this problem, we sought to develop a genetic screening platform that could be used in the mammalian nervous system in a fashion similar to the way that genetic screens are used in simpler model organisms such as Caenorhabditis elegans and Drosophila melanogaster. We term this methodology SLIC (for synthetic lethal in the CNS). SLIC is composed of four parts: a short hairpin RNA (shRNA) library, stereotaxic intracranial injection, mouse models of disease, and DNA sequencing (Fig. 2). The principle of synthetic lethality is that factors that are dispensable in a healthy cell are rendered essential in a diseased cell; these factors thus define the pathways responsible for increased cellular vulnerability in that disease. To identify synthetic lethal interactions, SLIC uses a pooled screen approach that was pioneered in yeast (11) and that has been adapted to identify tumor suppressors and oncogenes in mammalian cancer models (12–15). In SLIC, each neuron in a defined brain region receives a single perturbation, in this case a lentivirus encoding an shRNA, allowing many perturbations to be tested simultaneously in a single mouse, as opposed to one mouse being used as a screening vehicle for a single perturbation as in traditional knockout or knockdown experiments. Based on test injections into the mouse striatum, we calculate that we can transduce up to 2.8 × 10⁵ striatal cells per mouse (SI Appendix, Fig. S1) and that over 80% of viral-transduced cells are neurons (SI Appendix, Fig. S2).Open in a separate windowFig. 2.Synthetic lethal in the CNS (SLIC) screening. (Top) Lentiviral knockdown libraries are injected into the striatum, such that each neuron or glial cell receives a distinct element (schematized by different colors). Lentivirus integrates into the cell’s genome and expresses either a cDNA or shRNA. (Bottom) After incubation in vivo, cells that have received a synthetic lethal hit will die (*) and the representation of these library elements will be lost (an event that can be revealed by sequencing of all of the lentiviruses still present in the brain). As neurons in adult animals do not proliferate, the change in abundance detected by sequencing is due to cell loss. When injections are performed in a paired fashion, comparing disease model mice to wild-type littermates, genes that cause synthetic lethality only in combination with a disease-causing mutation can be identified.For our first SLIC screen, we focused on Huntington’s disease, as it is an ideal test case for a genetic screen: the disease is monogenic, affects defined cell populations in an age-dependent manner, and several mouse models have been created that display minimal cell loss. This latter feature is particularly advantageous to our screening scheme, as synthetic lethal screens require a mild phenotype around which to screen for an enhanced phenotype. We performed a SLIC screen in the R6/2 Huntington’s disease model line (16), seeking genes that, when knocked down, would enhance mutant huntingtin toxicity. We used 95 shRNAs in our screen (Dataset S9), including 1 positive control shRNA, 7 negative control shRNAs, and 87 shRNAs targeting 28 candidate genes. The 28 candidates included 19 genes identified in our cell-type–specific aging study, six genes previously linked to Huntington’s disease, and three randomly chosen genes (see Dataset S9 for details). Viral pools were injected bilaterally into mouse striata of disease model and control littermates at 6 wk of age. Genomic DNA was then harvested at a control time point (2 d postinjection, once the lentivirus has integrated into the cellular DNA but before significant target protein knockdown), and two experimental time points at which we expected to observe synthetic lethality (4 and 6 wk postinjection). Finally, shRNA-coding sequences were amplified and sequenced to assess the relative abundance of the 95 shRNAs in the pool.To validate our SLIC approach, we first focused on the positive and negative control shRNAs included in our screen. Comparison of viral shRNA representation in the wild-type control (nonmodel) mouse striatal samples at 4 wk versus 2 d revealed that the positive control lentivirus, carrying an shRNA targeting the Psmd2 gene product (a proteasomal subunit, depletion of which is expected to lead to cell death), was greatly reduced in representation, whereas negative controls, which have no expected target in the mouse genome, were not reduced in representation (Fig. 3A). We next asked which shRNAs were lost to a higher degree from R6/2 Huntington’s disease model mice versus control littermates at the 4 and 6 wk experimental time points. This comparison revealed that all shRNAs targeting Gpx6, a glutathione peroxidase that by homology is predicted to detoxify H₂O₂ to water, demonstrated synthetic lethality with mutant huntingtin. Using a gene-scoring scheme that incorporates the synthetic lethality ranks of the top two shRNAs for each gene, Gpx6 was the top-ranking gene at both the 4 and 6 wk time points (Fig. 3 B and C; Datasets S10–S12; SI Appendix). We also observed several shRNAs that were depleted in wild-type cells, and thus presumably are deleterious even in healthy cells. Importantly, these shRNAs were lost to an equal or lesser extent in R6/2 brains, demonstrating that the Gpx6 interaction is specific and not a consequence of cells in the disease model showing increased vulnerability to a broad spectrum of potential cell stressors (Fig. 3B).Open in a separate windowFig. 3.SLIC screening in mouse models of Huntington’s disease. (A) Changes in control shRNA representation, as determined by sequencing, in the striatum of wild-type animals at 4 and 6 wk after injection compared with the control 2-d time point. A negative number reflects loss versus the control time point. Error bars reflect ±SD across shRNAs. (B) shRNA representation at the first SLIC HD time point. The log₂ fold changes in shRNA representation at 4 wk compared with the control 2-d time point for the HD model (R6/2, y axis) are plotted versus the log₂ fold changes at the same two time points for wild-type controls (WT, x axis). The positive control targeting the Psmd2 gene product is not plotted for the purposes of scaling. Diagonal line represents equal representation for visual reference (x = y). Genes causing synthetic lethality are expected to be offset to the right of the diagonal. Gpx6-targeting shRNAs are denoted in red. (C) SLIC results for shRNAs showing synthetic lethality in the HD model. The y axes show the percentage of depletion of the indicated shRNAs in the HD model compared with wild-type controls, where “depletion” is defined as “the log₂ fold change in representation between early (2 d) and late (4 wk, Left; 6 wk, Right) time points in each genotype” and “% depletion” is defined as “the difference in depletion in the disease model relative to wild-type controls {[(WT-R6/2)/WT] × 100}.” Sample values were averaged across four replicates. Multiple annotations of the same target gene on the x axis represent distinct shRNAs targeting these genes. Gpx6-targeting shRNAs are denoted in red.Because little is known about Gpx6 function and expression, we next assessed its distribution across brain region and age. We found Gpx6 to be highly expressed in the olfactory bulb, striatum, and frontal cerebral cortex (SI Appendix, Fig. S3) and, confirming our TRAP results, observed that Gpx6 expression increases with age (SI Appendix, Fig. S4). Because our screen identified Gpx6 as a gene that, when reduced, enhances mutant huntingtin toxicity, we next asked whether overexpression of this gene product would have a therapeutic effect on phenotype progression in a Huntington’s disease mouse model. We overexpressed Gpx6 in the striatum of the R6/2 model and congenic wild-type control mice via adeno-associated viral (AAV) transduction starting at 6 wk of age. Two weeks after viral injection, we observed a dramatic rescue of open-field and rota-rod motor behavior in R6/2 mice, but no effect on motor behavior in wild-type control mice (Fig. 4A and SI Appendix, Fig. S6). Finally, analysis of a molecular marker of Huntington’s disease progression, loss of DARPP-32 striatal expression (17), revealed that Gpx6 overexpression also increases DARPP-32 expression in the R6/2 model (Fig. 4B). Thus, Gpx6 overexpression protects against both behavioral and molecular phenotypes associated with disease progression in this mouse model.Open in a separate windowFig. 4.(A) Rescue of open-field motor behavior in Huntington’s disease model mice overexpressing Gpx6. Huntington’s disease model mice (R6/2) or wild-type (WT) congenic controls were injected in the striatum bilaterally with Gpx6 or control (EGFP-L10a construct expressing; EGFP-L10a is most prominently observed in the nucleolus) AAV9 virus at 6 wk of age. After 2 wk of recovery, motor function was assessed by open field assay. Average performance is plotted ±SEM for each data point, reflecting total distance in centimeters traveled during a 1-h interval (R6/2 + Gpx6, n = 10; R6/2+ control, n = 10; WT + Gpx6, n = 12; WT + control, n = 11). R6/2 + Gpx6 vs. R6/2 + control, P value = 0.0165; WT + Gpx6 vs. WT + control, P value = 0.7826 (no significance). (B) Increased DARPP-32 expression in Huntington’s disease model mice overexpressing Gpx6. Huntington’s disease model mice (R6/2) or wild-type (WT) congenic controls were unilaterally injected with control (EGFP-L10a construct; left hemisphere) or Gpx6 overexpressing (right hemisphere) AAV9 virus at 6 wk of age. After 2 wk of recovery, mice were killed and brain tissue was processed for indirect immunofluorescent staining. (Top) Representative images of R6/2 mice injected with Gpx6 and control AAV9. (Bottom) Quantitation of images (mean pixel intensity across imaging field) from equivalent points in the dorsal striatum; P value = 0.0026. No significant difference between control and Gpx6-injected hemispheres was observed in wild-type congenic controls (SI Appendix, Fig. S5). A.U. signifies arbitrary fluorescence units.Our data demonstrate that our SLIC methodology can be used to identify genes that display synthetic lethality in combination with a disease-associated mutation. This screen tested 95 shRNAs using just a single mouse brain hemisphere per replicate, demonstrating its power and scaleability. In principle, both genome-wide overexpression and knockdown screens can be conducted in the mammalian central nervous system using SLIC. Our screen used constitutively expressing lentiviruses to target all striatal cells, but increased cellular specificity could be achieved through use of conditional systems such as the TVA/EnvA system (18). The gene identified in our screen as enhancing the toxicity of mutant huntingtin when knocked down, Gpx6, is particularly appealing given the abundant literature linking oxidative stress to Huntington’s disease pathophysiology (19), a study reporting that mice deficient in cellular glutathione peroxidase show enhanced vulnerability to a chemical model of Huntington’s disease (20), a recent study identifying glutathione peroxidase activity as a suppressor of mutant Huntingtin toxicity in yeast (21), and a study reporting Gpx6 protein levels to be altered in the striatum of human Huntington’s disease patients (22). Furthermore, blood–brain barrier-penetrant, orally active glutathione peroxidase-mimicking hydrogen peroxide scavengers have been identified (23, 24). Given that Gpx6 interacts genetically with the mutant huntingtin, and that we find that it is up-regulated with age, our screen suggests that increases to reactive oxygen species (hydrogen peroxide in particular) contribute to the enhancement of mutant huntingtin toxicity that is seen with advancing age.     

α-Synuclein phosphorylation at serine 129 occurs after initial protein deposition and inhibits seeded fibril formation and toxicity

α-Synuclein (α-syn) phosphorylation at serine 129 (pS129–α-syn) is substantially increased in Lewy body disease, such as Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). However, the pathogenic relevance of pS129–α-syn remains controversial, so we sought to identify when pS129 modification occurs during α-syn aggregation and its role in initiation, progression and cellular toxicity of disease. Using diverse aggregation assays, including real-time quaking-induced conversion (RT-QuIC) on brain homogenates from PD and DLB cases, we demonstrated that pS129–α-syn inhibits α-syn fibril formation and seeded aggregation. We also identified lower seeding propensity of pS129–α-syn in cultured cells and correspondingly attenuated cellular toxicity. To build upon these findings, we developed a monoclonal antibody (4B1) specifically recognizing nonphosphorylated S129–α-syn (WT–α-syn) and noted that S129 residue is more efficiently phosphorylated when the protein is aggregated. Using this antibody, we characterized the time-course of α-syn phosphorylation in organotypic mouse hippocampal cultures and mice injected with α-syn preformed fibrils, and we observed aggregation of nonphosphorylated α-syn followed by later pS129–α-syn. Furthermore, in postmortem brain tissue from PD and DLB patients, we observed an inverse relationship between relative abundance of nonphosphorylated α-syn and disease duration. These findings suggest that pS129–α-syn occurs subsequent to initial protein aggregation and apparently inhibits further aggregation. This could possibly imply a potential protective role for pS129–α-syn, which has major implications for understanding the pathobiology of Lewy body disease and the continued use of reduced pS129–α-syn as a measure of efficacy in clinical trials.

Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) are both associated with underlying Lewy body disease, which represents the second most common neurodegenerative disorder after Alzheimer’s disease (1, 2). The neuropathological hallmark of Lewy body disease is the intracellular aggregation of the protein α-synuclein (α-syn) into spherical cytoplasmic inclusions, termed Lewy bodies, but are also observed in neuronal processes as Lewy neurites (LNs) (3).α-Syn is thought to play a central role in the pathobiology of Lewy body disease. Single-point mutations and genetic modifications affecting α-syn expression—through duplications, triplications, or polymorphisms in its promoter—have been linked to both idiopathic and familial forms of Lewy body disease (4–6). Nevertheless, neuropathological studies utilizing pan–α-syn antibodies, recognizing both physiological and pathological forms of the protein, do not consistently report a relationship between the load of Lewy body pathology and clinical disease severity (2). To reconcile the apparent importance of α-syn in Lewy body disease with the difficulty relating Lewy body burdens in the brain to phenotypic severity, continued research has focused on the identification of particularly disease-relevant forms of α-syn. α-Syn undergoes various posttranslational modifications (PTMs)—including acetylation, nitration, ubiquitination, and glycosylation and phosphorylation at serine 129 (pS129)—increases from ∼4% under physiological conditions to 90% in Lewy body disease, suggesting it is associated with the disease state (7–9).Previous studies have reported that pS129 enhances intracellular aggregate formation in SH-SY5Y cells (10), and mediates cell death through activation of the unfolded protein response pathway (11). Furthermore, studies in rodent models have suggested that pS129 exacerbates the rate of pathological protein aggregation and deposition, with subsequent negative effects on neuronal functioning (12). However, these studies are counterbalanced by others reporting a potentially neuroprotective function of phosphorylation in animal models (13, 14) and cellular model systems (15). Additionally, studies have reported neutral findings regarding pS129 modification as neither enhancing nor diminishing cellular toxicity and α-syn aggregation (16, 17). Despite the uncertain pathogenic role of pS129 in Lewy body disease, antibodies against pS129 are widely used, based on the putative view that they label a species of α-syn that is particularly disease-relevant. These studies often employ pS129–α-syn as a marker of the abundance of protein inclusions to stage disease severity and evaluate the relationship between its abundance and important clinical or pathological variables, such as disease duration, phenotypic severity, or cell loss (18). Such studies typically identify that pS129 abundance throughout the brain correlates with disease severity (19–21), though it remains uncertain whether phosphorylation precedes protein aggregation or occurs secondarily to deposition of nonphosphorylated α-syn, and whether pS129 is a key driver of pathogenicity or simply a useful marker of a neurodegenerative process (22, 23). Therefore, although there is a substantial literature on pS129 in Lewy body disease, there is continued controversy regarding its potential contribution to disease states, with numerous studies reporting discordant findings. Despite contradictory findings regarding the disease-relevance of pS129, it is widely viewed as a particularly disease-associated modification, thus necessitating further research to address its importance for Lewy body disease.To address the key questions regarding the pathogenic relevance of pS129–α-syn, the present study aimed to undertake a comprehensive and multidisciplinary project to address this important and pressing question. The key aim of the study was to better understand the role of pS129 in the natural history of Lewy body disease, by determining when pS129 occurs in the development of α-syn aggregates and how it affects the aggregation-propensity and cytotoxicity of α-syn     

Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expansion in the number of polyglutamine-encoding CAG repeats in the gene that encodes the huntingtin (htt) protein. A property of the mutant protein that is intimately involved in the development of the disease is the propensity of the glutamine-expanded protein to misfold and generate an N-terminal proteolytic htt fragment that is toxic and prone to aggregation. Intracellular antibodies (intrabodies) against htt have been shown to reduce htt aggregation by binding to the toxic fragment and inactivating it or preventing its misfolding. Intrabodies may therefore be a useful gene-therapy approach to treatment of the disease. However, high levels of intrabody expression have been required to obtain even limited reductions in aggregation. We have engineered a single-domain intracellular antibody against htt for robust aggregation inhibition at low expression levels by increasing its affinity in the absence of a disulfide bond. Furthermore, the engineered intrabody variable light-chain (V(L))12.3, rescued toxicity in a neuronal model of HD. We also found that V(L)12.3 inhibited aggregation and toxicity in a Saccharomyces cerevisiae model of HD. V(L)12.3 is significantly more potent than earlier anti-htt intrabodies and is a potential candidate for gene therapy treatment for HD. To our knowledge, this is the first attempt to improve affinity in the absence of a disulfide bond to improve intrabody function. The demonstrated importance of disulfide bond-independent binding for intrabody potency suggests a generally applicable approach to the development of effective intrabodies against other intracellular targets.     

NAD+ supplementation reduces neuroinflammation and cell senescence in a transgenic mouse model of Alzheimer’s disease via cGAS–STING

Alzheimer''s disease (AD) is a progressive and fatal neurodegenerative disorder. Impaired neuronal bioenergetics and neuroinflammation are thought to play key roles in the progression of AD, but their interplay is not clear. Nicotinamide adenine dinucleotide (NAD⁺) is an important metabolite in all human cells in which it is pivotal for multiple processes including DNA repair and mitophagy, both of which are impaired in AD neurons. Here, we report that levels of NAD⁺ are reduced and markers of inflammation increased in the brains of APP/PS1 mutant transgenic mice with beta-amyloid pathology. Treatment of APP/PS1 mutant mice with the NAD⁺ precursor nicotinamide riboside (NR) for 5 mo increased brain NAD⁺ levels, reduced expression of proinflammatory cytokines, and decreased activation of microglia and astrocytes. NR treatment also reduced NLRP3 inflammasome expression, DNA damage, apoptosis, and cellular senescence in the AD mouse brains. Activation of cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) are associated with DNA damage and senescence. cGAS–STING elevation was observed in the AD mice and normalized by NR treatment. Cell culture experiments using microglia suggested that the beneficial effects of NR are, in part, through a cGAS–STING-dependent pathway. Levels of ectopic (cytoplasmic) DNA were increased in APP/PS1 mutant mice and human AD fibroblasts and down-regulated by NR. NR treatment induced mitophagy and improved cognitive and synaptic functions in APP/PS1 mutant mice. Our findings suggest a role for NAD⁺ depletion-mediated activation of cGAS–STING in neuroinflammation and cellular senescence in AD.

Alzheimer’s disease (AD) is the most feared neurodegenerative disease and is characterized by progressive cognitive impairment associated with extensive accumulation of amyloid β-peptide (Aβ) plaques and tau neurofibrillary tangles in vulnerable brain regions (1, 2). There are no available treatments. Neuroinflammation, mitochondrial dysfunction, and cellular senescence have been recognized as key drivers of AD (3–6). Microglia are the primary innate immune cells in the brain. Although accumulating evidence challenges the simplified M1-M2 phenotypes of microglia, the classification is still widely in use as microglia can be protective (M2) and detrimental (M1) under different circumstances (7, 8). The detrimental microglia are activated by Aβ and produce interleukin (IL)-1β, TNF-α, and other proinflammatory molecules. The protective microglia secrete the anti-inflammatory cytokines IL-4 and IL-10 (8).The inflammatory response is one of the hallmarks of cellular senescence (9). The senescence-associated secretory phenotype (SASP) includes cessation of cell division and the production of proinflammatory cytokines (10). The SASP is highly correlated with neuroinflammation and has been documented in the brains of AD mouse models (11). During normal aging, the number of senescent cells in tissues increases significantly (10), and evidence suggests that microglia, astrocytes, and oligodendrocyte progenitor cells can become senescent in AD (5, 11, 12). Moreover, senolytic treatments can preserve cognitive function in AD mice (11). However, the mechanisms that result in neuroinflammation and cell senescence in AD remain unclear.Nicotinamide adenine dinucleotide (NAD⁺) plays a central role in cellular metabolism and is also critical for maintaining mitochondrial homeostasis and genome integrity (13). Emerging evidence has identified lower levels of NAD⁺ in affected tissues in many neurodegenerative diseases, including AD (13–16). Supplementation with the NAD⁺ precursor nicotinamide riboside (NR) efficiently increases NAD⁺ and can have beneficial effects on many AD features in mouse models (14). The cyclic GMP-AMP synthase (cGAS)–STING (stimulator of interferon genes) DNA-sensing pathway detects the presence of cytosolic DNA and triggers expression of inflammatory genes that lead to senescence or to the activation of defense mechanisms (17–20). Here, we explore the mechanisms by which NAD⁺ supplementation reduces neuroinflammation and cell senescence in AD. Our results suggest that the cGAS–STING pathway is a therapeutic target for AD.     

Genome-wide screen identifies curli amyloid fibril as a bacterial component promoting host neurodegeneration

Growing evidence indicates that gut microbiota play a critical role in regulating the progression of neurodegenerative diseases such as Parkinson’s disease. The molecular mechanism underlying such microbe–host interaction is unclear. In this study, by feeding Caenorhabditis elegans expressing human α-syn with Escherichia coli knockout mutants, we conducted a genome-wide screen to identify bacterial genes that promote host neurodegeneration. The screen yielded 38 genes that fall into several genetic pathways including curli formation, lipopolysaccharide assembly, and adenosylcobalamin synthesis among others. We then focused on the curli amyloid fibril and found that genetically deleting or pharmacologically inhibiting the curli major subunit CsgA in E. coli reduced α-syn–induced neuronal death, restored mitochondrial health, and improved neuronal functions. CsgA secreted by the bacteria colocalized with α-syn inside neurons and promoted α-syn aggregation through cross-seeding. Similarly, curli also promoted neurodegeneration in C. elegans models of Alzheimer’s disease, amyotrophic lateral sclerosis, and Huntington’s disease and in human neuroblastoma cells.

Neurodegenerative diseases are characterized by protein misfolding and aggregation, leading to the formation of amyloid fibril enriched in β-sheet structures. Such protein aggregates trigger proteotoxicity, overwhelm the chaperone and degradation machineries, and eventually cause neuronal death (1). For example, Parkinson’s disease (PD) is associated with the intracellular aggregation of α-synuclein (α-syn) into Lewy bodies and Lewy neurites, which causes the degeneration of mostly dopaminergic (DA) neurons in the substantia nigra (2). The loss of DA neurons leads to decreased dopamine signaling in the striatum, which results in impaired motor functions in PD patients. α-syn is a small (140–amino acid) protein made of an N-terminal domain, a non–A-β component domain that is the fibrilization core, and a carboxyl-terminal region. Missense mutations in the N-terminal domain, such as A30P, G46K, and A53T, result in autosomal dominant familial PD by producing mutant proteins that are more prone to misfolding and aggregation than the wild-type proteins (3). Mutant α-syn proteins form toxic β-sheet–like oligomers that cause mitochondrial dysfunctions, oxidative stress, disruption in calcium homeostasis, and neuroinflammation, which all lead to neurodegeneration (2). Effective therapeutic intervention that prevents α-syn aggregation is currently missing.Studies in the last few years have suggested that the gut microbiota may play an important role in the pathogenesis of neurodegenerative diseases (4). For example, antibiotic treatment ameliorates the pathophysiology of PD mice, and microbial recolonization after the treatment restored the PD symptoms (5). Colonization of α-syn–overexpressing mice with microbiota from PD patients exacerbated the physical impairments compared to transplantation of microbiota from healthy donors (5). In addition to animal models, clinical studies have also provided evidence for a microbiota–gut–brain link in PD. Gastrointestinal dysfunction was frequently found in PD patients (6), and infection with Helicobacter pylori has been linked with disease severity and progression (7). Sequencing of the fecal samples of PD patients revealed changes in the gut bacterial composition (e.g., increased Lactobacillaceae) compared to healthy individuals (8, 9). Similar to PD, gut bacteria in mouse models of Alzheimer’s disease (AD) promoted amyloid pathology (10), and altered gut microbiome composition was also observed in AD patients (11).Despite the growing connection between the disturbed gut microbiota and the development of neurodegenerative diseases, mechanistic understanding of the communication between the bacteria and the nervous system is limited. Most theories focus on the neurodegenerative effects of the systemic inflammation and neuroinflammation caused by the abnormal microbiota. Whether bacteria proteins or metabolites can directly act on the host neurons to module the progression of neurodegeneration induced by α-syn or A-β proteotoxicity is unclear. This limitation is largely due to the lack of a simple model that allows systematic tests of individual bacterial components for their neuronal effects.To address the problem, we employed a Caenorhabditis elegans model of PD that expressed the human α-syn proteins in C. elegans neurons to investigate the mechanisms of microbial regulation on PD. Because C. elegans uses bacteria as its natural diet and can be easily cultivated under monoaxenic conditions, it has emerged as an important organism to model microbe–host interaction. In fact, alteration of the bacterial genome affected the development, metabolism, and behavior of C. elegans (12, 13). Several studies also showed that certain bacterial metabolites could influence neurodegeneration in C. elegans (14–16).In this study, we screened all nonessential Escherichia coli genes for their effects on PD pathogenesis by feeding individual E. coli knockout mutants to PD C. elegans and assessing the severity of neurodegeneration. This screen identified 38 E. coli genes whose deletion led to amelioration of PD symptoms. These genes fall into distinct genetic pathways including curli formation, lipopolysaccharide (LPS) production, lysozyme inhibition, adenosylcobalamin synthesis, and oxidative stress response, suggesting that diverse bacteria components could promote neurodegeneration. As an example, we focused on the role of bacterial curli amyloid fibril on PD and found that deleting the curli genes csgA and csgB in the E. coli genome reduced α-syn–induced cell death, restored mitochondrial health, and improved neuronal functions. Using antibody staining and biochemical analysis, we showed that CsgA promoted α-syn aggregation and that removing curli in the bacteria diet enabled proteasome-dependent degradation of α-syn. Importantly, we demonstrated in vivo colocalization and cross-seeding between bacteria-derived CsgA and α-syn in dopaminergic neurons; this cross-seeding significantly enhanced α-syn aggregation. Moreover, we extended our findings into C. elegans models of AD, amyotrophic lateral sclerosis (ALS), and Huntington’s disease (HD) and into human neuroblastoma SH-SY5Y cells. Overall, our studies indicate that bacterial components, such as curli, can have direct neurodegenerative effects.     

Calcium homeostasis modulator 1 (CALHM1) is the pore-forming subunit of an ion channel that mediates extracellular Ca2+ regulation of neuronal excitability

Extracellular Ca(2+) (Ca(2+)(o)) plays important roles in physiology. Changes of Ca(2+)(o) concentration ([Ca(2+)](o)) have been observed to modulate neuronal excitability in various physiological and pathophysiological settings, but the mechanisms by which neurons detect [Ca(2+)](o) are not fully understood. Calcium homeostasis modulator 1 (CALHM1) expression was shown to induce cation currents in cells and elevate cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) in response to removal of Ca(2+)(o) and its subsequent addback. However, it is unknown whether CALHM1 is a pore-forming ion channel or modulates endogenous ion channels. Here we identify CALHM1 as the pore-forming subunit of a plasma membrane Ca(2+)-permeable ion channel with distinct ion permeability properties and unique coupled allosteric gating regulation by voltage and [Ca(2+)](o). Furthermore, we show that CALHM1 is expressed in mouse cortical neurons that respond to reducing [Ca(2+)](o) with enhanced conductance and action potential firing and strongly elevated [Ca(2+)](i) upon Ca(2+)(o) removal and its addback. In contrast, these responses are strongly muted in neurons from mice with CALHM1 genetically deleted. These results demonstrate that CALHM1 is an evolutionarily conserved ion channel family that detects membrane voltage and extracellular Ca(2+) levels and plays a role in cortical neuronal excitability and Ca(2+) homeostasis, particularly in response to lowering [Ca(2+)](o) and its restoration to normal levels.     

Therapeutic silencing of mutant huntingtin with siRNA attenuates striatal and cortical neuropathology and behavioral deficits

Huntington's disease (HD) is a neurodegenerative disorder caused by expansion of a CAG repeat in the huntingtin (Htt) gene. HD is autosomal dominant and, in theory, amenable to therapeutic RNA silencing. We introduced cholesterol-conjugated small interfering RNA duplexes (cc-siRNA) targeting human Htt mRNA (siRNA-Htt) into mouse striata that also received adeno-associated virus containing either expanded (100 CAG) or wild-type (18 CAG) Htt cDNA encoding huntingtin (Htt) 1-400. Adeno-associated virus delivery to striatum and overlying cortex of the mutant Htt gene, but not the wild type, produced neuropathology and motor deficits. Treatment with cc-siRNA-Htt in mice with mutant Htt prolonged survival of striatal neurons, reduced neuropil aggregates, diminished inclusion size, and lowered the frequency of clasping and footslips on balance beam. cc-siRNA-Htt was designed to target human wild-type and mutant Htt and decreased levels of both in the striatum. Our findings indicate that a single administration into the adult striatum of an siRNA targeting Htt can silence mutant Htt, attenuate neuronal pathology, and delay the abnormal behavioral phenotype observed in a rapid-onset, viral transgenic mouse model of HD.     

Preclinical Detection of Alpha-Synuclein Seeding Activity in the Colon of a Transgenic Mouse Model of Synucleinopathy by RT-QuIC

In synucleinopathies such as Parkinson’s disease (PD) and dementia with Lewy body (DLB), pathological alpha-synuclein (α-syn) aggregates are found in the gastrointestinal (GI) tract as well as in the brain. In this study, using real-time quaking-induced conversion (RT-QuIC), we investigated the presence of α-syn seeding activity in the brain and colon tissue of G2-3 transgenic mice expressing human A53T α-syn. Here we show that pathological α-syn aggregates with seeding activity were present in the colon of G2-3 mice as early as 3 months old, which is in the presymptomatic stage prior to the observation of any neurological abnormalities. In contrast, α-syn seeding activity was not detectable in 3 month-old mouse brains and only identified at 6 months of age in one of three mice. In the symptomatic stage of 12 months of age, RT-QuIC seeding activity was consistently detectable in both the brain and colon of G2-3 mice. Our results indicate that the RT-QuIC assay can presymptomatically detect pathological α-syn aggregates in the colon of G2-3 mice several months prior to their detection in brain tissue.     

Biomaterials for Drugs Nose–Brain Transport: A New Therapeutic Approach for Neurological Diseases

In the last years, neurological diseases have resulted in a global health issue, representing the first cause of disability worldwide. Current therapeutic approaches against neurological disorders include oral, topical, or intravenous administration of drugs and more invasive techniques such as surgery and brain implants. Unfortunately, at present, there are no fully effective treatments against neurodegenerative diseases, because they are not associated with a regeneration of the neural tissue but rather act on slowing the neurodegenerative process. The main limitation of central nervous system therapeutics is related to their delivery to the nervous system in therapeutic quantities due to the presence of the blood–brain barrier. In this regard, recently, the intranasal route has emerged as a promising administration site for central nervous system therapeutics since it provides a direct connection to the central nervous system, avoiding the passage through the blood–brain barrier, consequently increasing drug cerebral bioavailability. This review provides an overview of the nose-to-brain route: first, we summarize the anatomy of this route, focusing on the neural mechanisms responsible for the delivery of central nervous system therapeutics to the brain, and then we discuss the recent advances made on the design of intranasal drug delivery systems of central nervous system therapeutics to the brain, focusing in particular on stimuli-responsive hydrogels.     

Soluble α-synuclein–antibody complexes activate the NLRP3 inflammasome in hiPSC-derived microglia

Parkinson’s disease is characterized by accumulation of α-synuclein (αSyn). Release of oligomeric/fibrillar αSyn from damaged neurons may potentiate neuronal death in part via microglial activation. Heretofore, it remained unknown if oligomeric/fibrillar αSyn could activate the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome in human microglia and whether anti-αSyn antibodies could prevent this effect. Here, we show that αSyn activates the NLRP3 inflammasome in human induced pluripotent stem cell (hiPSC)-derived microglia (hiMG) via dual stimulation involving Toll-like receptor 2 (TLR2) engagement and mitochondrial damage. In vitro, hiMG can be activated by mutant (A53T) αSyn secreted from hiPSC-derived A9-dopaminergic neurons. Surprisingly, αSyn–antibody complexes enhanced rather than suppressed inflammasome-mediated interleukin-1β (IL-1β) secretion, indicating these complexes are neuroinflammatory in a human context. A further increase in inflammation was observed with addition of oligomerized amyloid-β peptide (Aβ) and its cognate antibody. In vivo, engraftment of hiMG with αSyn in humanized mouse brain resulted in caspase-1 activation and neurotoxicity, which was exacerbated by αSyn antibody. These findings may have important implications for antibody therapies aimed at depleting misfolded/aggregated proteins from the human brain, as they may paradoxically trigger inflammation in human microglia.

Parkinson’s disease (PD) is characterized by accumulation of α-synuclein (αSyn; encoded by the SNCA gene) (1). Release of oligomeric/fibrillar αSyn from damaged neurons may potentiate neuronal cell death in part via microglial activation (2, 3). Moreover, misfolded proteins in general are thought to interact with brain microglia, triggering microglial activation that contributes to neurodegenerative disorders, although microglial phagocytosis may also initially clear aberrant proteins to afford some degree of protection (2, 4). Additionally, in Alzheimer’s disease (AD), amyloid-β peptide (Aβ) is thought to trigger similar processes in microglia (5–7); however, the mechanism for this trigger is still poorly understood.Microglial cells contribute to neuroinflammation, specifically that mediated by the inflammasome. In particular, the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome has been associated with several neurodegenerative disorders, although other types of inflammation may also be important in this regard (8). The NLRP3 inflammasome is a multiprotein complex that responds to cell stress and pathogenic stimuli to promote activation of caspase-1, which in turn mediates maturation and release of proinflammatory cytokines, including interleukin-1β (IL-1β) and IL-18 (9–11). NLRP3 inflammasome activation is a two-step process, involving an initial priming step and a secondary trigger. Priming involves a proinflammatory stimulus, such as endotoxin, a ligand for Toll-like receptor 4 (TLR4), that increases the abundance of NLRP3 and promotes de novo synthesis of pro–IL-1β via nuclear factor κB (11). The secondary trigger promotes inflammasome complex assembly and caspase-1 activation that in turn mediates the cleavage of pro–IL-1β and subsequent release of mature IL-1β. There are various secondary triggers, including adenosine triphosphate (ATP), microparticles, and bacterial toxins, all of which somehow lead to mitochondrial damage and release of oxidized mitochondrial DNA (11). Neuroinflammation has been reported in both human PD and AD brains (12–15), and NLRP3 inflammasome activation in particular has been observed in mouse models of PD and AD (7, 16). Importantly, in these PD models, dopaminergic (DA) neurons in the substantia nigra are resistant to damage in NLRP3-deficient mice compared with wild-type (WT) mice (16). Interestingly, a recent report identified an NLRP3 polymorphism that confers decreased risk in PD (17). Several groups have reported that fibrillar αSyn can activate the NLRP3 inflammasome in mice and in human monocytes (18–22), but it remains unknown if human brain microglia can be activated in this manner. Critically, antibodies targeting misfolded proteins are being tested in human clinical trials for several neurodegenerative diseases, including AD and PD; however, it is still unclear how antibodies to αSyn might affect this inflammatory response. In this study, we characterized the response of human induced pluripotent stem cell (hiPSC)-derived microglia (hiMG) to oligomeric/fibrillar αSyn in vitro and in vivo, using engraftment of hiMG in humanized mice. We used these immunocompromised mice because they prevent human cell rejection and express three human genes that support human cell engraftment (23). We show that αSyn and, even more so, αSyn–antibody complexes activate the NLRP3 inflammasome. Moreover, this process is further sensitized by the presence of Aβ and its cognate antibodies. These observations are of heightened interest because recent studies have shown that both misfolded Aβ and αSyn are present in several neurodegenerative disorders such as AD and Lewy body dementia (LBD), a form of dementia that can occur in the setting of PD (24–26).